Project summary Sepsis is a clinical syndrome with life-threatening organ dysfunction that is caused by a dysregualted host response to infection. Lung is one of the first organs to fail during sepsis and contribute to high mortality. Lung endothelial dysfunction is a hallmark of sepsis-induced acute lung injury (ALI), where inflammation plays an important role. We have recently found a rise in the plasma cell-free RNA including miRNAs in sepsis. These extracellular (ex) RNAs are released from host cells as well as invading bacteria and closely correlated with sepsis severity. Plasma miRNA array identifies six miRNAs that are elevated in response to sepsis; four of them (miR-34a, -122, -145, -146a) induce marked cytokine production and complement activation in macrophages via the innate immune TLR7 signaling. Importantly, systemic inhibition of ex-miR-146a attenuates cytokine IL-6 production in the plasma and the lung tissue of septic animals. These preliminary data suggest an important role of ex-miRNAs in acute lung inflammation and possible lung injury in sepsis. This MIRA research program will test the hypothesis that ex-miRNA plays an important role in acute lung injury during polymicrobial sepsis. Specifically, the Program will address the following 3 key questions: 1) what is the role of ex-miRNAs in sepsis-induced ALI? 2) what are the underlying molecular mechanisms responsible for the ex-miRNA-mediated ALI in sepsis? 3) can we develop an anti-miRNA strategy to treat sepsis-induced ALI? To answer these key questions, the Program is structured with 3 projects. In Project-1, we will define the role of the target 4 ex-miRNAs in sepsis- induced ALI using anti-miR strategy and carefully evaluate the biological function of these ex- miRNAs and their carriers (exosomes and Ago-2) in inflammatory response. In Project-2, we will determine the underlying mechanisms by which miRNAs increase lung endothelial cell permeability with focus on cell junction protein VE-cadherin and Claudin-5. The impact of the ex-miRNAs on the cell-cell interaction between leukocytes and lung endothelial cells will also be examined. In Project-3, through a series of pre-clinical and proof-of-concept studies, we will assess the feasibility and efficacy of developing a new therapeutic strategy by targeting ex-miRNAs and their specific receptor TLR7 in the treatment of sepsis-induced ALI. The toxicity and safety of the anti-miRNAs and TLR7 antagonist will be carefully tested.